The present invention relates to methods for the detection or isolation of prion proteins by use of chaperones specifically binding to said proteins. The invention further relates to a method for in-vitro diagnosis of a transmissible spongiform encephalopathy and to pharmaceutical compositions, preferably for the prevention or treatment of said disease.
Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases such as scrapie of sheep, bovine spongiform encephalopathy (BSE) of cattle and Creutzfeldt-Jakob disease (CJD) of man (34). Infectious preparations derived from infected brains are resistant to ultraviolet and ionizing radiation as well as other procedures which inactivate nucleic acids indicating that nucleic acids may not be required for infectivity. Purification of infectious preparations from brains revealed the presence of a protein required for infectivity (36). These experimental observations led to the xe2x80x98protein onlyxe2x80x99 hypothesis, which proposes that proteinaceous infectious particles (xe2x80x98prionsxe2x80x99) are responsible for the transmission of TSEs (3, 4, 36). Prions consist mainly of a protease resistant protein designated PrPSc (prion protein, xe2x80x98Scxe2x80x99 for scrapie), a posttranslationally modified form of the proteinase K sensitive host encoded PrPc (xe2x80x98cxe2x80x99 for cellular) (8, 9, 11, 34). Both isoforms share the same amino acid sequence, but differ in their secondary structure (31, 42). Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy revealed a significantly higher xcex2-sheet content for PrPSc as compared to a high xcex1-helix content in PrPc (17, 31, 38). Structural predictions of PrPc led to a model which proposed that four domains between amino acid residues 109 to 122, 129 to 141, 178 to 191 and 202 to 218 form xcex1-helices (24). It has been suggested that prion propagation involves the conversion of xcex1-helical domains in PrPc into xcex2-sheets in PrPSc (26, 30, 31). The in vitro conversion of PrPc into PrPSc was demonstrated employing a proteinase K resistance assay (28). A modified model was recently suggested according to which PrPc must be partially unfolded and refolded into PrPSc under the direction of an oligomeric PrPSc seed (29). This model provides explanations for scrapie species barriers (27) and strain-specific properties of prions (7). In addition, experiments employing transgenic mice led to the proposal that prion propagation requires a species-specific macromolecule designated xe2x80x98protein Xxe2x80x99 (43).
So far, there is no method described allowing the straightforward detection or isolation of natural prion proteins. The isolation of PrPc described in the prior art (31) is time consuming, ineffective and yields only minimum amounts of protein. The isolation of PrPsc described in the prior art (31, 35, 64) is also time consuming and ineffective and the purity of the PrPsc is speculative. Furthermore, up to now it was not possible to discriminate between the cellular isoform PrPc and the isoform PrPsc or PrP27-30, which is a prerequisite for the development of a simple and reliable assay for diagnosing a transmissible spongiform encephalopathy.
Tatzeh et al., PNAS USA 92 (1995), 2944-2984, investigate proteins that might feature in the conversion of the cellular prion protein (PrPc) into the scrapie isoform (PrPsc). It was found that in scrapie-infected N2a cells the expression and subcellular translocation of specific heat shock proteins was altered. However, said document does not disclose that chaperones like the heat shock proteins specifically bind to prion proteins and, thus, can be used to detect or isolate prion proteins.
Therefore, the technical problem underlying the present invention is to provide a simple method for the efficient isolation of prion proteins and the detecion of said proteins, preferably in a way that allows for discrimination between different isoforms of PrP.
The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
Thus, the present invention relates to a method for the detection of a prion protein comprising the steps of:
(a) contacting a probe suspected to contain a prion protein with a chaperone, and
(b) determining whether a prion protein binds to the chaperone.
In addition, the present invention relates to a method for the isolation of a prion protein comprising the steps of:
(a) contacting a probe containing a prion protein with a chaperone, and
(b) isolating the chaperone-bound protein from the chaperone.
When carrying out experiments in order to identify proteins capable of interacting with PrPc it was surprisingly found that chaperones are capable of specifically binding to prion proteins. The specificity of the observed in vivo interactions was confirmed by in vitro binding studies employing recombinant prion proteins. Mapping of the interaction site between the molecular chaperones and PrPc was performed using recombinant prion GST-fusion peptides. The results show that a GST-PrPc fusion protein binds specifically to Hsp60 in an S. cerevisiae environment as well as in vitro. The Hsp60 family is one of the best characterized members of the molecular chaperones which mediate ATP-dependent folding of polypeptide chains (13, 18, 22, 23) and which are widely distributed and conserved between prokaryotes and mammals. Human Hsp60 (544 amino acids) is proposed to form tetradecameric complexes in vivo as shown in the crystal structure of the prokaryotic homologue GroEL (10). The cDNAs isolated by a two-hybrid screen in S. cerevisiae (15, 19, 21) encode N-terminally truncated proteins of 399, 317 and 246 amino acids in length, comprising at least in part the apical domain of the Hsp60 monomer. This apical domain contains several amino acid residues which specifically mediate peptide binding in the case of GroEL (14). Specificity of the PrPc/Hsp60 interaction in vivo was confirmed employing the xe2x80x98false baitsxe2x80x99 LexA-bicoid and LexA-NFI/CTF2 as well as authentic LexA and LexA-GST. The interaction was confirmed in vitro using recombinant GST-PrPc and recombinant full-length Hsp60 as well as GroEL. This result shows that the PrPc/Hsp60 interaction does not involve additional factors and that thus, chaperones can be used for the detection and isolation of prion proteins. The recombinant rPrP27-30 (47) represents the proteinase K sensitive isoform of the proteinase K resistant core PrP27-30 isolated from scrapie preparations. The results of the in vitro interaction between rPrP27-30 and Hsp60 reveal that the core region of PrP (amino acids 90 to 231) is sufficient for binding to Hsp60.
Identification of the interaction site between amino acid 180 and amino acid 210 by mapping of PrPc peptides showed that binding of Hsp60 to PrPc occurs within a highly conserved region of the prion protein containing amino acids 180, 198, 200 and 210. Mutation of these residues segregate with inherited prion diseases in humans (33). In addition, the chaperone-binding fragment GST::P180-210 contains at least in part the two putative xcex1-helical domains H3 (amino acids 178 to 191) and H4 (amino acids 202 to 218) (24). The conversion of xcex1-helical regions into xcex2-sheets of PrP are thought to be responsible for PrPSc formation. There are several possibilities to suggest a possible physiological relevance of the Hsp60/PrP interaction. (i) Hsp60 might be involved in the propagation of PrPSc as has been shown for the interaction of the yeast prion-like factor [psi+] with the molecular chaperone Hsp104 (12, 50). Based on studies with transgenic mice, it has been suggested recently that a species-specific macromolecule, designated xe2x80x98protein Xxe2x80x99, participates in prion formation (43). Protein X was proposed to function as a molecular chaperone facilitating the transformation of PrP isoforms. This unknown factor xe2x80x98Xxe2x80x99 might in fact be Hsp60. (ii) Alternatively, Hsp60 could prevent aggregation of PrPc to PrPSc amyloids e.g. by trapping misfolded forms of PrPc.
More recent data suggested that so-called xe2x80x9cchemical chaperonesxe2x80x9d such as glycerol, trimethylamine N-oxide (TMAO), and dimethylsulfoxide (DMSO) interfere with PrPSc formation by stabilizing the xcex1-helical conformation of PrPc. (67)
The detection or isolation of prion proteins by the methods of the invention relates to recombinantly produced prion proteins or prion proteins from natural sources. Prion proteins can be extracted from natural sources, for example, by the method described in (31, 64); involving suspending tissue in sucrose, homogenization and clarification by centrifugation.
Contacting the probe suspected to contain a prion protein with a chaperone can be carried out by known methods, for example, with the chaperone being in solution or being immobilized, for example on a matrix such as a gel or a resin for chromatography (66).
Contacting of the probe with the chaperone and analyzing of the complex chaperone-prion protein can also be carried out as described in the Examples below.
Suitable chaperones which specifically bind to prion proteins can be inter alia determined by the person skilled in the art by assaying the binding of a particular chaperone to prion proteins as described in the Examples, below.
In one preferred embodiment of the method of the invention, a fragment, analogue or derivative of said chaperone is used which is still capable of binding the prion protein.
As used herein, the term xe2x80x9cderivativexe2x80x9d refers to such derivatives which may be prepared from the functional groups which occur at side chains on the residues or the N- or C-terminus groups, by means known in the art.
The term xe2x80x9cfragmentxe2x80x9d relates to any fragment of the chaperone which still has the capability to interact with the prion protein and such a fragment can be prepared by techniques known to the person skilled in the art.
In another preferred embodiment of the invention, the chaperone used in the method for detection and/or isolation of a prion protein is Hsp60 or GroEL.
In a further preferred embodiment the chaperone is a recombinant protein, i.e., the chaperone is produced by recombinant DNA technology, namely by expression from a cloned DNA sequence.
In a still further preferred embodiment, the chaperone is part of a fusion protein, which can comprise, besides the chaperone a protein or preferably, a protein domain which confers to the fusion protein a specific binding capacity. Preferably, the recombinant chaperone is fused to glutathione-S-transferase.
Any prion protein, isoform, fragment or derivative of such prion protein or mixture of said substances can be detected or isolated by the method of the invention as long as it is capable of being bound by the chaperone. In a preferred embodiment the prion protein to be detected or isolated is the prion protein PrPc and/or an isoform of PrPc. Preferably the prion protein isoform is the isoform PrPsc or a fragment or derivative thereof.
In a further preferred embodiment the prion protein is the processed form PrPc23-231 comprising amino acids 23 to 231 of PrPsc and/or the isoform PrPsc is the N-terminally truncated derivative PrP27-30 or a fragment thereof.
As already stated above, for determining whether a prion protein was bound to a chaperone, the chaperone can be in solution or be attached to a solid phase.
Following incubation of both participants in solution, the interaction can be proved by co-immunoprecipitation (51) followed by Western Blotting (44) employing a PrP specific antibody as described, for example, in (20), a chaperone-specific antibody or an antibody directed against one of the Tags, i.e. GST-antibody, FLAG-antibody, BTag-antibody, antibody directed against the calmodulin binding protein, the S-peptide, the maltose-binding-protein, oligohistidine and the green fluorescent protein (GFP). Furthermore, the interaction can, for example, be proved by (i) crosslinking employing reagents such as dimethylsuberimidate (52), (ii) by affinity chromatography (66) by adding the immobilized ligand directed against one of the tags fused to one of the two partners (Criss-Cross interactions), or (iii) by analyzing the complex by a non-denaturing polyacrylamide gel (53) or by a size exclusion chromatography which is mostly HPLC/FPLC (54).
In a preferred embodiment, the chaperone is in solution and detectably labelled. The person skilled in the art will know suitable labels or will be able to ascertain such labels using routine experimentation. Preferably the detectable label is selected from a radioisotope, a fluorescent compound, a colloidal metal, a chemiluminescent compound, a bioluminescent compound, a phosphorescent compound or an enzyme.
Alternatively, the chaperone is bound to a solid phase for the detection and/or isolation of a prion protein. Suitable materials are known to the person skilled in the art and include, for example, a gel or a resin (Sepharose, agarose, nitrocellulose, dynabeads(copyright), polystyrene etc.).
In a preferred embodiment, the solid phase is a matrix comprising glutathione, such as glutathione-sepharose. The protein domain used for binding of the chaperone to a matrix can also be an oligohistidine (55), Calmodulin binding peptide (CBP) (56), S-peptide (ribonuclease A) (57), FLAG (58), green-fluorescent protein (GFP, 65), BTag (59), or maltose-binding-protein (MBP; 61). The tagged chaperone can be immobilized to gluthathione, IMAC-Ni2+. Calmodulin, S-protein 104 aa (57), anti-FLAG-antibodies, anti GFP-antibodies, BTag-antibodies (59) or maltose (60).
Alternatively, coupling of the chaperone itself by the fusion protein can be done via thiol-groups of non-oxidized cysteins or, alternatively, via free lysine or xcex1-amino groups to cyanogen bromide agarose or a-hydroxy succinimide activated agarose (63).
Regarding the interaction of the prion proteins with chaperones where one of the compounds is immobilized, the interaction can be determined by IASYS (FISONS). Protein-protein interactions can be detected and measured by biosensors, which use the evanescent field to probe biomolecular mass and concentration close to the probe surface. Alternatively, such interaction can be determined by far western blot/affinity blot (62): the prion-protein either tagged or untagged or the chaperone either tagged or untagged are blotted onto a membrane such as nitrocellulose or PVDF. The other interaction partner either the tagged/untagged prion protein or the tagged/untagged chaperone in solution is incubated with the protein associated membrane. Interaction is confirmed by addition of an antibody directed against the protein in solution itself or the tag fused to the protein (62).
In a still further preferred embodiment of the method of the invention for isolating prion proteins, the chaperone is part of a matrix contained within an affinity chromatography column (63, 66) and step (b) is modified in such a way that
(i) the probe suspected to contain the prion protein is passed through the column,
(ii) after washing, the prion protein is eluted from the column, optionally by a change in pH or ionic strength and collected; and
(iii) optionally the collected prion protein is further purified.
By this kind of affinity chromatography which is, for example, described in (55, 63, 66), impurities contained in the prion protein preparation are passed through the column. The prion protein(s) will be bound to the column by the chaperone. Suitable conditions for allowing the specific binding of the prion protein to the column and for eluting the prion protein from the gel can be determined by the person skilled in the art and are, for example, described in (47, 48).
In an alternative embodiment, the isolation of the prion protein is carried out as a batch process according to standard procedures or, for example, by using a modified version of the procedure described in the Examples, below, wherein instead of the prion protein, the chaperone is attached to glutathione-Sepharose beads, for example, gluthathione-Sepharose 4B beads.
Prion proteins isolated and purified according to the method of the invention can be used, for example, as immunogen for raising antibodies, as active component of pharmaceutical compositions or for the development of diagnostic assays, such as ELISA.
The probe can be obtained from various organs, preferable from tissue, for example brain, ileum, cortex, dura mater, purkinje cells, lymphnodes, nerve cells, spleen, tonsils, muscle cells, placenta, pancreas, eyes, backbone marrow or peyer""sche plaques from a body fluid, preferably from blood, cerebrospinal fluid, semen or milk.
As is evident from the results presented in Example 6, binding of the chaperone GroEL is stronger to rPrP27-30 compared to PrPc (see lane 3 of FIG. 2B versus lane 2 of FIG. 2C). These results were confirmed by further titration experiments with GroEl and Hsp60 (data not shown). Thus, determining the strength of binding of a chaperone with the prion protein in a probe by comparing it with the strength of binding of the same chaperone with PrPsc (or rPrP27-30) and PrPc standards allows the determination of whether a prion protein indicative for transmissible spongiform encephalopathy (TSE) is contained in a sample.
Accordingly, a further preferred embodiment of the invention relates to a method for the in-vitro diagnosis of a transmissible spongiform encephalopathy, wherein step (b) is modified in such a way that the differences in binding of the chaperone to PrPc and an isoform of PrPc, respectively, preferably PrPsc, are used to determine whether an isoform of PrPc is present in the probe or not.
The present invention furthermore provides a complex of the chaperone and a prion protein and, in addition, a composition for the detection and/or isolation of a prion protein comprising a chaperone as defined above.
Furthermore, the present invention relates to a diagnostic composition comprising the chaperones as defined above. Such compositions may contain additives commonly used for diagnostic purposes. Said compositions can be used for the diagnosis of transmissible spongiform encephalopathies by applying the approach described above, wherein a probe taken from a body is incubated with a chaperone and the strength of binding of the chaperone to the prion protein contained in the probe is determined. In the case that brain is used as a probe, diagnosis is often carried out post mortem but is, in certain cases, also possible on the living organism (biopsy). In the case that blood, milk or cerebrospinal fluid is used as a probe, diagnosis is possible for living individuals.
In another embodiment, the present invention relates to a pharmaceutical composition comprising a chaperone as defined above or, alternatively, comprising a substance that inactivates said chaperone. Such compositions can optionally comprise pharmaceutically acceptable carriers.
Since, for example, chaperones like Hsp60 are assumed to be capable of preventing the aggregation of PrPc to PrPsc, it might be possible to block the conversion of the isoform PrPc into the prion associated isoform PrPsc by administration of such chaperones which specifically bind prion proteins and, thus, to prevent or treat transmissible spongiform encephalopathy.
On the other hand, it might be possible that chaperones are involved in the transformation of PrPc to PrPsc. Thus, blocking such transformation by the administration of agents which specifically inactivate such chaperones which specifically interact with prion proteins could also be helpful for the treatment or prevention of transmissible spongiform encephalopathies. Such substances can be selected by the person skilled in the art by routine experimentation and include ligands that bind to the chaperone, thus preventing the interaction of the chaperone with the prion protein. Examples of such ligands are antibodies, preferably monoclonal antibodies, or a fragment of a protein which a domain responsible for binding to the chaperone, e.g. a fragment of PrPc containing amino acids 180 to 210.
Preferably, said compositions are used for the prevention or treatment of transmissible spongiform encephalopathy, for example, Scrapie, bovine spongiform encephalopathy (BSE), Creutzfeld-Jacob Disease (CJD), Gerstmann-Strxc3xa4uxcex2ler-Scheinker-Syndrome (GSS), Kuru, fatal familial insomnia (FFI) or transmissible mink encephalopathy (TME).